Peptides for inhibiting HPV E6-proteins

ABSTRACT

The present invention relates to peptides suited for inhibiting HPV-E6 proteins, DNAs encoding them and the use of both, particularly for eliminating HPV tumor cells.

The present invention relates to peptides which are suited to inhibitHPV-E6 proteins, DNAs coding for them and the use of both, particularlyfor eliminating HPV-positive cells, e.g. HPV tumor cells.

Human papilloma viruses (HPVs) are closely related with the developmentof carcinomas. The involvement of HPVs in the development of thecervical carcinoma is well characterized, and various findings refer tothe fact that HPVs play a causal part in the etiology of this carcinoma.On a molecular level, sequences of HPVs, particularly HPV 16 and 18, aredetectable in about 95% of the cervical carcinoma biopsies. In thisconnection, the HPV DNA is usually present in integrated form in thegenome of the tumor cells. It frequently comprises deletions and/orrearrangements, which never refer to the early HPV genes, namely the E6and E7 genes. These genes code for the HPV proteins E6 and E7 which areresponsible for the formation and manifestation of HPV carcinomas. Inthis connection, the HPV proteins have a synergistic effect. On theother hand, there are references to the effect that they also displayopposite activities. For example, the E7 protein induces apoptosis,whereas the E6 protein inhibits it. This is due to the fact that the E6protein binds to p53 directly or indirectly, i.e. via ubiquintin-proteinligase E6-AP, and inhibits it, so that p53 can no longer display itsapoptosis-inducing activity. The E6 protein also has an anti-apoptoticactivity independent of p53. The genes of the E6 and E7 proteins areexpressed via polycistronic mRNAs, the transcription being controlled bya common promoter. Experiments of inhibiting the latter and/or E6-/E7mRNAs are known. These experiments resulted in a parallel inhibition ofthe growth of HPV tumor cells. However, elimination of such cells, whichis most desired, could not be achieved by this.

Therefore, it is the object of the present invention to provide aproduct by which HPV tumor cells can be eliminated.

According to the invention this is achieved by the subject mattersdefined in the claims.

The applicant found that HPV-E6 proteins bind to short peptides. Hescreened a randomized oligopeptide library which comprises randomlygenerated peptide sequences with a “peptide aptamer system” in which theHPV-E6 protein was used as a screening sample. He found that shortpeptides, particularly those listed in Table 1, bind HPV-E6 proteins. Healso found that these peptides are suited to inhibit activities ofHPV-E6 proteins, e.g. their anti-apoptotic activity. In addition, heobserved that an elimination of HPV-positive cells, particularly HPVtumor cells, can be achieved by this inhibition.

According to the invention, the applicant's insights are used to providea peptide which is selected from the peptides listed in Table 1, whereinthe peptide may comprise a sequence modification of up to 40%,particularly 20% and most particularly 10%.

E61-1.pep: NH₂-GALVHKLFSQ TSGSCLVCIS-COOH (SEQ ID NO: 1) E61-2.pep:NH₂-LDVLGCLVRR LGVVLVGLH-COOH (SEQ ID NO: 2) E61-3.pep: NH₂-CYVECGCEVLTALVNGVRVL-COOH (SEQ ID NO: 3) E61-5.pep: NH₂-GVGGLCSCAS CVSEDFYASV-COOH(SEQ ID NO: 4) E61-7.pep: NH₂-IDLLRRLGSQL HLLLVSVGG-COOH (SEQ ID NO: 5)E61-8.pep: NH₂-LAVLLNGYTR AIVGISFGGW-COOH (SEQ ID NO: 6) E61-9c.pep:NH₂-LCTMCATVFR PLLVWFWSIW-COOH (SEQ ID NO: 7) E61-10.pep: NH₂-QLLLDLLLGSYEGMSLTSSP-COOH (SEQ ID NO: 8) E61-11.pep: NH₂-SRSNALHTLD VLLGGT-COOH(SEQ ID NO: 9) E61-12.pep: NH₂-GGAVYLCDAG CCFYCCGCSG-COOH (SEQ ID NO:10) E61-13.pep: NH₂-CLELFDDLFL ALSLLLLVGG-COOH (SEQ ID NO: 11)E61-14.pep: NH₂-PLCRTCLIES AVLIQLSRL-COOH (SEQ ID NO: 12) E61-15.pep:NH₂-VFSGVYYAEF VFAASAGGTP-COOH (SEQ ID NO: 13) E61-16.pep:NH₂-MAPVGAGRPC CTVCFLTARF-COOH (SEQ ID NO: 14) E61-17.pep:NH₂-LSMLLFAAKL PVAVLCSWQA-COOH (SEQ ID NO: 15) E61-19.pep:NH₂-LVGRVRIGVS VFIRGGRLL-COOH (SEQ ID NO: 16) E61-20.pep: NH₂-LFDIFRLCAQPVLVHGHTRV-COOH (SEQ ID NO: 17)

Peptides according to the invention are suited to bind HPV-E6 proteinsand inhibit them as regards their activities, e.g. as regards theiranti-apoptotic activity.

The expression “HPV-E6 proteins” comprises an E6 protein of any HPVtype, particularly of HPV1, 5, 6, 11, 16, 18, 31, 33 or 35. An E6protein can have a wild-type sequence or a sequence differing therefromby one or several amino acids. Furthermore, it may be present inshortened form, i.e. it is only available as the fragment which isnecessary to bind to p53, ubiquintin-protein ligase E6-AP or anotherbinding participant of the E6 protein. The fragment can also be presentin multiple copies within a polypeptide molecule. An E6 protein or afragment thereof can also be present in the form of a fusion protein.

Peptides according to the invention can be provided by common methods inwhich peptides are tested as regards their binding to HPV-E6 proteins.Such methods are e.g. the “peptide aptamer” or “bacteriophage display”method. It is favorable to use the “peptide aptamer” method which isdescribed in the examples and which is a modification of theabove-mentioned method.

Peptides according to the invention can be present as such or incombination with other substances, e.g. (poly)peptides. The combinationmay consist in linking the peptides according to the invention with the(poly)peptides via linkers, e.g. disulfide bridges. The peptidesaccording to the invention can also be fused with the (poly)peptides, sothat the peptides according to the invention are present in the form offusion (poly)peptides. For example, leader peptides, such as penetratinfrom Drosophila antennapedia or VP22 from HSV1, which support theabsorption of the peptides according to the invention in cells, offerthemselves as (poly)peptides for fusion (poly)peptides. On the otherhand, polypeptides which are linked with the peptide according to theinvention via linkers, can be e.g. carrier proteins, such astransferrin, which are not considered foreign by the body. Severalpeptides according to the invention can also be simultaneously presentin combination with an above-mentioned substance.

A further subject matter of the present invention relates to a nucleicacid, particularly a DNA which codes for a peptide according to theinvention. Such a DNA can be present in vectors, particularly expressionvectors. A person skilled in the art is familiar with examples thereof.In the case of an expression vector for E. coli, these are e.g. pGEMEX,pUC derivatives, pGEX-2T, pET3b and pQE-8. For the expression in yeast,e.g. pY100 and Ycpad1 have to be mentioned while e.g. pKCR, PEFBOS, cDM8or pCEV4 have to be indicated for the expression in animal cells. Thebaculovirus expression vector pAcSGHisNT-A is especially suited for theexpression in insect cells. It is also possible to use viruses, e.g.adenovirus, vaccinia virus, adeno-associated virus (AAV) orretroviruses, such as MoMuLV, HaMuSV, MuMTV, RSV or GaIV, for theexpression in animal cells.

The person skilled in the art is familiar with suitable cells to expressa DNA according to the invention, which is present in an expressionvector. Examples of such cells comprise the E. coli strains HB101, DH1,x1776, JM101, JM109, BL21 and SG13009, the yeast strain Saccharomycescerevisiae and the animal cells L, NIH 3T3, FM3A, CHO, COS, Vero andHeLa as well as the insect cells sf9.

The person skilled in the art knows in which way the DNA according tothe invention has to be inserted in an expression vector. He is alsofamiliar with the fact that this DNA can be inserted in combination witha DNA coding for another peptide or polypeptide, so that the DNAaccording to the invention can be expressed in the form of a fusionpolypeptide.

In addition, the person skilled in the art knows conditions of culturingtransformed or transfected cells. He is also familiar with methods ofisolating and purifying the peptide or fusion polypeptide, which isexpressed by the DNA according to the invention.

A further subject matter of the present invention relates to an antibodydirected against an above peptide or fusion polypeptide. Such anantibody can be prepared by common methods. It may be polyclonal ormonoclonal. For its preparation it is favorable to immunizeanimals—particularly rabbits or chickens for a polyclonal antibody andmice for a monoclonal antibody—with an above (fusion) polypeptide orwith fragments thereof. Further “boosters” of the animals can beeffected with the same (fusion) polypeptide or with fragments thereof.The polyclonal antibody may then be obtained from the animal serum oregg. For the preparation of the monoclonal antibody, animal spleen cellsare isolated and fused with myeloma cells.

A further subject matter of the present invention relates to apharmaceutical composition which contains one or several peptidesaccording to the invention and/or DNAs encoding them as well asconventional auxiliary agents. The auxiliary agents used can be e.g.carriers, binders, blasting agents, lubricants, solvents, solution aids,release accelerators, release retarders, emulsifiers, stabilizers, etc.Such a composition can be used as usual, e.g. orally or parenterally.The suitable dosage is determined in the individual case as usual.

A further subject matter of the present invention relates to adiagnostic composition which contains one or several peptides accordingto the invention. HPV-E6 proteins can be detected by such a composition.This can be used to detect HPV-associated diseases, such as HPVinfections, HPV dysplasias of HPV carcinomas. Such a detection comprisese.g. (a) collection of a cell sample from a patient, (b) contacting ofthe cell sample with a peptide according to the invention underconditions permitting the specific binding of the peptide to an HPV-E6protein, and (c) detection of the peptide. This detection can be made bystandard methods. For example, the peptides according to the inventioncan be present in the liquid phase or be bound to a solid carrier and belabeled in different ways. The person skilled in the art is familiarwith suitable markers and labeling methods. The peptides can also bedetected by antibodies according to the invention. The latter are alsosuited to control the therapeutic course of an HPV-associated diseasetreated with peptides according to the invention.

By means of the present invention it is possible to bind HPV-E6 proteinsand inhibit their activities, particularly their anti-apoptoticactivity. This serves for achieving the elimination of HPV-positivecells, particularly HPV tumor cells. HPV-positive cells can also bediagnosed. Therefore, the present invention is suitable to takediagnostic and therapeutic steps against HPV-associated diseases,particularly HPV infections, HPV dysplasias or HPV carcinomas. Inaddition, the peptides according to the invention and/or DNAs encodingthem represent a basis for the development of fully new activesubstances against the above diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the modified “peptide aptamer” system in S. cerevisiae byway of diagram. This system comprises three components: (1) the targetprotein (E6) which is fused with a heterologous DNA binding domain(GAL4BD), (2) a peptide having randomized amino acid sequence, which isfused to a transcriptional activation domain (GAL4AD), and (3) a stablyintegrated selection gene (prototrophic selection markers, such asADE2), which has the recognition sequence for the DNA binding domain inits promoter. A synthetic transcription factor forms by interactionbetween peptide and target protein. It binds to the recognition sequencein the promoter region of the selection gene by the transactivationdomain and stimulates the transcription of the selection gene by thetransactivation domain. Under selection conditions (e.g.adenine-negative media) only the yeast cells form colonies which expressa peptide with affinity for the target protein (TrxA=i E. colithioreduxin A; TAG=influenza virus HA-Tag; NLS=nuclear localizationsignal).

FIG. 2 shows the analysis of HPV16-E6-binding peptides in the “peptideaptamer” system. By screening about 2×10⁶ yeast cells, 15 positiveclones are isolated (cf. Table 1, E61-1.pep-E61.-17.pep). Replicaplatings of the yeast colonies (master plate above: 1-15=positiveclones; K=randomly selected control clone) are carried out underselection for ADE 2(GAL4-BS in context of the GAL2 promoter), HIS3(GAL4BS on the basis of the GAL1 promoter) and URA3 (GAL4-BS on thebasis of the SPO13 promoter).

The following examples explain the invention.

EXAMPLE 1 Screening for HPV-E6 Protein-binding Peptides

A method is used which is derived from the known “peptide aptamer”system. The method is shown in FIG. 1 by way of diagram.

For the screening for HPV-E6 protein-binding peptides, a randomizedoligopeptide expression library is established for 20 amino acid-longpeptides having random sequence (complexity about 2×10⁸ differingpeptides). Codons are defined by the sequence NNK (N=G, A, T or C; K=Gor C). They code for all of the 20 amino acids, but only result in onestop codon. A yeast expression vector, pADTrx, is used as the expressionvector. It contains E. coli Trx A (thioreduxin protein) from pTrx(Invitrogen company) and GAL4AD as well as the ADH promoter from pAS2and/or pGAD424 (Clontech company). The peptides are expressed on thebasis of the active loop of Trx. This comprises the followingadvantages:

the presentation of the peptides outwards is ensured on the basis of theTrx loop,

conformally restricted peptides can expose amino acids outwards whichare possibly folded inwards in flexible peptides in the intracellularenvironment,

conformally restricted peptides can be highly affine peptides, whichhave the potential of acting as efficient protein inhibitors in vivo aswell.

A yeast strain, KF-1, is also used. It originates from the yeast strainPJ69-4A (cf. James et al., Genetics 144 (1996), 1425) and permits theanalysis of three selection markers: ADE2, HIS3 and URA3. Each of thethree selection markers is under the transcriptional control of GAL4binding sites on the basis of various promoters. The URA3 selectionmarker is regulated by the SPO13 promoter which originates from theyeast strain MaV103 (cf. Vidal et al., Proc. Natl. Acad. Sci. USA (93),10315-10320), contains a negatively regulatory element and can beactivated by strong protein-protein interactions. The yeast strain alsocontains the E. coli β-galactosidase (-Gal) gene as further marker,whose activity can easily be quantified and enables an evaluation of thein vivo binding activity of the peptide to the E6 protein. Theactivation of the HIS3 gene can also be quantified by titration using3′AT-(3-amino-1, 2, 4-triazole).

The HPV16-E6 protein is subjected to screening using the above system.For this purpose, it is provided in the form of an expression vectorcoding for it. pPC97 (cf. Vidal et al., above) serves as a basic vectorin which the coding sequence of an HPV16-E6 protein is inserted. 15clones are isolated from about 2×10⁶ yeast clones, which show growthunder ADE2 selection. Replica platings and the analysis of the threeselection markers reveal that 14 of the 15 clones show growth also underHIS3 selection (FIG. 2). In addition, 6 of the isolated clones showadditionally growth under URA3 selection, which refers to an especiallyhigh in vivo affinity of the corresponding peptide for the HPV-E6protein under the conditions employed herein.

For further control the corresponding peptide expression plasmids areisolated from the 15 clones and subjected to rescreening in yeast afteranother transformation. The rescreening shows a complete conformity withthe results from the above replica platings (FIG. 2). It turns out thatthe growth of the yeast depends on the binding of the peptides to theHPV16-E6 protein.

The peptides of the 15 clones are determined as regards their amino acidsequence. It is indicated in Table 1, E61-1.pep-E61-17.pep. Part of thepeptides show sequence homologies with the binding domains, related toone another, of the E6-binding proteins E6-AP, E6-BP and paxillin(LhXΦLs″-like motives, in which: h=Q, E or N; X=any amino acid;Φ=hydrophobic amino acid; s=small amino acid such as G or A and −=acidicamino acid). The other part of the E6-binding peptides have no obvioussequence similarities with respect to the above E6-binding proteins.However, their sequences can give references to formerly unknown E6interaction participants.

In another screening assay, two further clones are obtained. Their aminoacid sequence is indicated in Table 1, E61-19.pep and E61-20.pep.

EXAMPLE 2 Inhibition of the Anti-apoptotic Activity of HPV-E6 Proteinsby Peptides According to the Invention

The “VP 22 shuttle system” is used. It is based on the fact that theHSV1-VP 22 protein is absorbed by cells, i.e. can be used as a carrier.Fusion polypeptides from VP 22 and peptides of Table 1 according to theinvention are prepared. For this purpose, the expression vector pCEP4(Invitrogen company) is used. The DNA sequences of the above peptidesare inserted therein in phase with the DNA sequence of VP 22. In thisconnection, the peptides are expressed on the basis of the E. coli TrxAprotein. The resulting expression plasmids pCEP4/ E61-1.pep and/orpCEP4/E61-2.pep are transfected in HPV16-positive SiHa and/or CaSKicervical carcinoma cells. The morphology of the cells and their growthis examined by common methods at various times.

It turns out that the anti-apoptotic activity of HPV-E6 proteins can beinhibited by the peptides according to the invention. Furthermore, itshows that the apoptosis induced by HPV-E7 proteins is obtained. This isalso reflected in “colony formation assays” which show strong growthinhibition of the cells. Thus, a peptide according to the invention issuited to eliminate HPV-positive cells, particularly HPV tumor cells.

17 1 20 PRT Artificial Sequence Peptide for inhibiting HPV-E6 protein 1Gly Ala Leu Val His Lys Leu Phe Ser Gln Thr Ser Gly Ser Cys Leu 1 5 1015 Val Cys Ile Ser 20 2 19 PRT Artificial Sequence Peptide forinhibiting HPV-E6 protein 2 Leu Asp Val Leu Gly Cys Leu Val Arg Arg LeuGly Val Val Leu Val 1 5 10 15 Gly Leu His 3 20 PRT Artificial SequencePeptide for inhibiting HPV-E6 protein 3 Cys Tyr Val Glu Cys Gly Cys GluVal Leu Thr Ala Leu Val Asn Gly 1 5 10 15 Val Arg Val Leu 20 4 20 PRTArtificial Sequence Peptide for inhibiting HPV-E6 protein 4 Gly Val GlyGly Leu Cys Ser Cys Ala Ser Cys Val Ser Glu Asp Phe 1 5 10 15 Tyr AlaSer Val 20 5 20 PRT Artificial Sequence Peptide for inhibiting HPV-E6protein 5 Ile Asp Leu Leu Arg Arg Leu Gly Ser Gln Leu His Leu Leu LeuVal 1 5 10 15 Ser Val Gly Gly 20 6 20 PRT Artificial Sequence Peptidefor inhibiting HPV-E6 protein 6 Leu Ala Val Leu Leu Asn Gly Tyr Thr ArgAla Ile Val Gly Ile Ser 1 5 10 15 Phe Gly Gly Trp 20 7 20 PRT ArtificialSequence Peptide for inhibiting HPV-E6 protein 7 Leu Cys Thr Met Cys AlaThr Val Phe Arg Pro Leu Leu Val Trp Phe 1 5 10 15 Trp Ser Ile Trp 20 820 PRT Artificial Sequence Peptide for inhibiting HPV-E6 protein 8 GlnLeu Leu Leu Asp Leu Leu Leu Gly Ser Tyr Glu Gly Met Ser Leu 1 5 10 15Thr Ser Ser Pro 20 9 16 PRT Artificial Sequence Peptide for inhibitingHPV-E6 protein 9 Ser Arg Ser Asn Ala Leu His Thr Leu Asp Val Leu Leu GlyGly Thr 1 5 10 15 10 20 PRT Artificial Sequence Peptide for inhibitingHPV-E6 protein 10 Gly Gly Ala Val Tyr Leu Cys Asp Ala Gly Cys Cys PheTyr Cys Cys 1 5 10 15 Gly Cys Ser Gly 20 11 20 PRT Artificial SequencePeptide for inhibiting HPV-E6 protein 11 Cys Leu Glu Leu Phe Asp Asp LeuPhe Leu Ala Leu Ser Leu Leu Leu 1 5 10 15 Leu Val Gly Gly 20 12 19 PRTArtificial Sequence Peptide for inhibiting HPV-E6 protein 12 Pro Leu CysArg Thr Cys Leu Ile Glu Ser Ala Val Leu Ile Gln Leu 1 5 10 15 Ser ArgLeu 13 20 PRT Artificial Sequence Peptide for inhibiting HPV-E6 protein13 Val Phe Ser Gly Val Tyr Tyr Ala Glu Phe Val Phe Ala Ala Ser Ala 1 510 15 Gly Gly Thr Pro 20 14 20 PRT Artificial Sequence Peptide forinhibiting HPV-E6 protein 14 Met Ala Pro Val Gly Ala Gly Arg Pro Cys CysThr Val Cys Phe Leu 1 5 10 15 Thr Ala Arg Phe 20 15 20 PRT ArtificialSequence Peptide for inhibiting HPV-E6 protein 15 Leu Ser Met Leu LeuPhe Ala Ala Lys Leu Pro Val Ala Val Leu Cys 1 5 10 15 Ser Trp Gln Ala 2016 19 PRT Artificial Sequence Peptide for inhibiting HPV-E6 protein 16Leu Val Gly Arg Val Arg Ile Gly Val Ser Val Phe Ile Arg Gly Gly 1 5 1015 Arg Leu Leu 17 20 PRT Artificial Sequence Peptide for inhibitingHPV-E6 protein 17 Leu Phe Asp Ile Phe Arg Leu Cys Ala Gln Pro Val LeuVal His Gly 1 5 10 15 His Thr Arg Val 20

What is claimed is:
 1. A peptide selected from the following peptides:NH₂-GALVHKLFSQ TSGSCLVCIS-COOH (SEQ ID NO: 1) NH₂-LDVLGCLVRRLGVVLVGLH-COOH (SEQ ID NO: 2) NH₂-CYVECGCEVL TALVNGVRVL-COOH (SEQ ID NO:3) NH₂-GVGGLCSCAS CVSEDFYASV-COOH (SEQ ID NO: 4) NH₂-IDLLRRLGSQLHLLLVSVGG-COOH (SEQ ID NO: 5) NH₂-LAVLLNGYTR AIVGISFGGW-COOH (SEQ ID NO:6) NH₂-LCTMCATVFR PLLVWFWSIW-COOH (SEQ ID NO: 7) NH₂-QLLLDLLLGSYEGMSLTSSP-COOH (SEQ ID NO: 8) NH₂-SRSNALHTLD VLLGGT-COOH (SEQ ID NO: 9)NH₂-GGAVYLCDAG CCFYCCGCSG-COOH (SEQ ID NO: 10) NH₂-CLELFDDLFLALSLLLLVGG-COOH (SEQ ID NO: 11) NH₂-PLCRTCLIES AVLIQLSRL-COOH (SEQ IDNO: 12) NH₂-VFSGVYYAEF VFAASAGGTP-COOH (SEQ ID NO: 13) NH₂-MAPVGAGRPCCTVCFLTARF-COOH (SEQ ID NO: 14) NH₂-LSMLLFAAKL PVAVLCSWQA-COOH (SEQ IDNO: 15) NH₂-LVGRVRIGVS VFIRGGRLL-COOH (SEQ ID NO: 16) NH₂-LFDIFRLCAQPVLVHGHTRV-COOH and (SEQ ID NO: 17) and a peptide containing a sequencemodification of up to 40% thereof; wherein the peptide binds to HPV-E6protein.
 2. The peptide according to claim 1, wherein the peptide ispresent as a fusion polypeptide.
 3. The peptide according to claim 2,wherein the fusion polypeptide comprises a leader sequence.
 4. A DNAcoding for the peptide according to claim
 1. 5. An expression vectorcontaining the DNA according to claim
 4. 6. An antibody directed againstthe peptide according to claim
 1. 7. A pharmaceutical compositioncomprising an auxiliary agent and a peptide selected from the followingpeptides: NH₂-GALVHKLFSQ TSGSCLVCIS-COOH (SEQ ID NO: 1) NH₂-LDVLGCLVRRLGVVLVGLH-COOH (SEQ ID NO: 2) NH₂-CYVECGCEVL TALVNGVRVL-COOH (SEQ ID NO:3) NH₂-GVGGLCSCAS CVSEDFYASV-COOH (SEQ ID NO: 4) NH₂-IDLLRRLGSQLHLLLVSVGG-COOH (SEQ ID NO: 5) NH₂-LAVLLNGYTR AIVGISFGGW-COOH (SEQ ID NO:6) NH₂-LCTMCATVFR PLLVWFWSIW-COOH (SEQ ID NO: 7) NH₂-QLLLDLLLGSYEGMSLTSSP-COOH (SEQ ID NO: 8) NH₂-SRSNALHTLD VLLGGT-COOH (SEQ ID NO: 9)NH₂-GGAVYLCDAG CCFYCCGCSG-COOH (SEQ ID NO: 10) NH₂-CLELFDDLFLALSLLLLVGG-COOH (SEQ ID NO: 11) NH₂-PLCRTCLIES AVLIQLSRL-COOH (SEQ IDNO: 12) NH₂-VFSGVYYAEF VFAASAGGTP-COOH (SEQ ID NO: 13) NH₂-MAPVGAGRPCCTVCFLTARF-COOH (SEQ ID NO: 14) NH₂-LSMLLFMKL PVAVLCSWQA-COOH (SEQ IDNO: 15) NH₂-LVGRVRIGVS VFIRGGRLL-COOH (SEQ ID NO: 16) NH₂-LFDIFRLCAQPVLVHGHTRV-COOH and (SEQ ID NO: 17) and a peptide containing a sequencemodification of up to 40% thereof; wherein the peptide binds to HPV-E6protein.
 8. The composition according to claim 7, wherein the peptide ispresent as a fusion polypeptide.
 9. The composition according to claim8, wherein the fusion polypeptide comprises a leader sequence.
 10. Amethod of treating a subject comprising administering to said subject apharmaceutically effective amount of the peptide according to claim 1,wherein said peptide inhibits the function of said HPV-E6 protein. 11.The method according to claim 10, wherein the peptide eliminatesHPV-positive cells.
 12. The method according to claim 11, wherein theHPV-positive cells originate from an HPV-associated disease.
 13. Themethod according to claim 12, wherein the HPV-associated disease isselected from the group consisting of an HPV infection, an HPVdysplasia, and an HPV carcinoma.
 14. The method according to claim 10,wherein HPV is selected from the group consisting of HPV1, HPV5, HPV6,HPV11, HPV16, HPV18, HPV31, HPV33, and HPV35.
 15. A DNA coding for thepeptide according to claim
 2. 16. A DNA coding for the peptide accordingto claim
 3. 17. An antibody directed against the peptide according toclaim
 2. 18. An antibody directed against the peptide according to claim3.
 19. The peptide according to claim 1, which is selected from thefollowing peptides: NH₂-GALVHKLFSQ TSGSCLVCIS-COOH (SEQ ID NO: 1)NH₂-LDVLGCLVRR LGVVLVGLH-COOH (SEQ ID NO: 2) NH₂-CYVECGCEVLTALVNGVRVL-COOH (SEQ ID NO: 3) NH₂-GVGGLCSCAS CVSEDFYASV-COOH (SEQ IDNO: 4) NH₂-IDLLRRLGSQL HLLLVSVGG-COOH (SEQ ID NO: 5) NH₂-LAVLLNGYTRAIVGISFGGW-COOH (SEQ ID NO: 6) NH₂-LCTMCATVFR PLLVWFWSIW-COOH (SEQ IDNO: 7) NH₂-QLLLDLLLGS YEGMSLTSSP-COOH (SEQ ID NO: 8) NH₂-SRSNALHTLDVLLGGT-COOH (SEQ ID NO: 9) NH₂-GGAVYLCDAG CCFYCCGCSG-COOH (SEQ ID NO:10) NH₂-CLELFDDLFL ALSLLLLVGG-COOH (SEQ ID NO: 11) NH₂-PLCRTCLIESAVLIQLSRL-COOH (SEQ ID NO: 12) NH₂-VFSGVYYAEF VFAASAGGTP-COOH (SEQ IDNO: 13) NH₂-MAPVGAGRPC CTVCFLTARF-COOH (SEQ ID NO: 14) NH₂-LSMLLFAAKLPVAVLCSWQA-COOH (SEQ ID NO: 15) NH₂-LVGRVRIGVS VFIRGGRLL-COOH (SEQ IDNO: 16) NH₂-LFDIFRLCAQ PVLVHGHTRV-COOH and (SEQ ID NO: 17).
 20. A methodof treating a subject comprising administering to said subject apharmaceutically effective amount of the pharmaceutical compositionaccording to claim 7, wherein said peptide of said pharmaceuticalcomposition inhibits the function of an HBV-E6 protein.
 21. A method forgenerating a peptide that binds to HPV-E6 protein, the method comprisingthe steps: a) generating multiple forms of peptides; b) testing forbinding between the peptides and HPV-E6 protein; and c) determining thesequence of a tested peptide that binds to the HPV-E6 protein.
 22. Amethod as described in claim 21, wherein the HPV protein is from HPV1,5, 6, 11, 16, 18, 31, 33, or
 35. 23. A method as described in claim 21,wherein the HPV protein is a fragment that binds p53 orubiquitin-protein ligase E6-AP.
 24. A method as described in claim 21,wherein the peptides are generated and displayed for binding to theHPV-E6 protein by a peptide aptamer or bacteriophage display method. 25.A peptide that binds to HPV-E6 protein, prepared by the method of claim21.
 26. A peptide that binds to HPV-E6 protein, prepared by the methodof claim 22.